Dopaminergic neuronal survival-promoting factors and uses thereof

ABSTRACT

In general, the invention features substantially purified MANF and substantially purified nucleic acids encoding the same. The invention also features a pharmaceutical composition that includes MANF and a pharmaceutically-acceptable excipient, methods for treatment of a neurodegenerative disease, methods for improving dopaminergic neuronal survival during or following cell transplantation, methods for production of neurons for transplantation, and methods for identifying compounds that modulate or mimic MANF&#39;s biological activity.

CROSS-REFERENCE

This application is a continuation application of U.S. application Ser.No. 10/102,265, filed Mar. 20, 2002, which claims benefit of andpriority to U.S. provisional application Ser. No. 60/277,516, filed Mar.20, 2001, both of which are incorporated herein by reference in theirentirety and to which applications we claim priority under 35 USC §§119,120.

BACKGROUND OF THE INVENTION

The invention relates to compositions and methods for increasing thesurvival of neurons.

The growth, survival, and differentiation of neurons in the peripheraland central nervous systems (PNS and CNS, respectively) are dependent,in part, on target-derived, paracrine, and autocrine neurotrophicfactors. Conversely, the lack of neurotrophic factors is thought to playa role in the etiology of neurodegenerative diseases such as Parkinson'sdisease, Alzheimer's disease, and amyotrophic lateral sclerosis (ALS orLou Gehrig's disease). In neuronal cell cultures, neurotrophic supportis provided by co-culturing with astrocytes or by providing conditionedmedium (CM) prepared from astrocytes. Astrocytes of ventralmesencephalic origin exert much greater efficacy in promoting thesurvival of ventral, mesencephalic dopaminergic neurons, compared withastrocytes from other regions of the CNS, such as the neostriatum andcerebral cortex. In chronic, mesencephalic cultures of 21 days in vitro(DIV) or longer, the percentage of dopaminergic neurons increases from20% to 60%, coincident with proliferation of a monolayer of astrocytes.In contrast, in conditions in which the proliferation of astrocytes wasinhibited, dopaminergic, but not GABAergic neurons, were almosteliminated from the cultures by 5 DIV. These results demonstrate theimportance of homotypically-derived astrocytes for the survival anddevelopment of adjacent dopaminergic neurons, and suggest thatmesencephalic astrocytes are a likely source of a physiological,paracrine neurotrophic factor for mesencephalic dopaminergic neurons.

The repeated demonstration that astrocytes secrete molecules thatpromote neuronal survival has made astrocytes a focus in the search fortherapeutics to treat neurodegenerative diseases. Many laboratories haveattempted to isolate astrocyte-derived neurotrophic factors, but havebeen hindered by a major technical problem: serum is an essentialcomponent of the medium for the optimal growth of primary astrocytes inculture, yet the presence of serum interferes with the subsequentpurification of factors secreted into the conditioned medium.

Thus, there is a need to identify and purify new neurotrophic factorsand to identify new methods to produce conditioned medium that arecompatible with protein isolation techniques.

SUMMARY OF THE INVENTION

We previously isolated a spontaneously immortalized type-1astrocyte-like cell line, referred to as ventral mesencephalic cellline-1 (VMCL-1). This cell line, deposited with the American TypeCulture Collection (ATCC; Manassas, Va.; ATCC Accession No: PTA-2479;deposit date: Sep. 18, 2000), was derived from the ventral mesencephalonand retained the characteristics of primary, type-1 astrocytes, butgrows robustly in a serum-free medium. The CM prepared from these cellscontains one or more neuronal survival factors that increase thesurvival of mesencephalic dopaminergic neurons at least 3-fold, andpromotes their development as well.

Using a multi-step purification process, we have identifiedarginine-rich protein (ARP) as a protein that co-purifies with thedopaminergic neuronal survival-promoting activity of VMCL-1 CM. As theprotein and the activity co-purified through five purification steps, weconclude that this protein is one of the factors in the VMCL-1 CM havingthe desired dopaminergic neuronal survival-promoting activity.

We have also discovered that ARP is produced in a previouslyunrecognized secreted form; we refer to this form as MANF (matureastrocyte-derived neurotrophic factor). MANF lacks the N-terminalarginine-rich portion of the protein, as is shown in FIG. 1 and SEQ IDNO: 3. Based on examination of the sequences, we believe that thissecreted form results from the cleavage of a previously unidentifiedsplice variant of ARP (ARPβ or pro-MANF), which has the sequence shownin SEQ ID NO: 2. MANF and pro-MANF, and biologically active analogs,derivatives, and fragments thereof, are collectively referred to as“MANF polypeptides.”

Accordingly, in a first aspect, the invention features a substantiallypurified MANF polypeptide. In one embodiment, the MANF polypeptide hasthe amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6, or 7. In anotherembodiment, the MANF polypeptide includes one or more conservative aminoacid substitutions relative to the amino acid sequence of SEQ ID NO: 2,3, 4, 5, 6, or 7, or is otherwise substantially identical to a proteinhaving one of these amino acid sequences.

In a second aspect, the invention features a substantially purifiedpolynucleotide encoding a MANF polypeptide. As described above, the MANFpolypeptide may have the amino acid sequence of SEQ ID NO: 2, 3, 4, 5,6, or 7, or may have one or more conservative amino acid substitutionsrelative to these amino acid sequences. In one embodiment, thepolynucleotide encodes a protein substantially identical to a proteinhaving the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6, or 7. Inanother embodiment, the polynucleotide consists of the sequence of SEQID NO: 9 or 10.

In a third aspect, the invention features an expression vector thatincludes the polynucleotide of the second aspect. The expression vectorcan be, for example, an adenoviral vector or a retroviral vector. In oneparticular embodiment, the polynucleotide is operably linked toregulatory sequences that allow for the expression of the polynucleotidein a neural cell.

In a fourth aspect, the invention features a pharmaceutical compositionthat includes: (i) a substantially purified MANF polypeptide; and (ii) acarrier that is pharmaceutically acceptable for administration to thecentral nervous system.

In a fifth aspect, the invention features a pharmaceutical compositionthat includes: (i) a substantially purified MANF polypeptide; (ii) apharmaceutically acceptable carrier; and (iii) a neural cell. The neuralcell can be, for example, a neuron, a neural stem cell, or a neuronalprecursor cell.

In a sixth aspect, the invention features a method for increasingsurvival of dopaminergic neurons, the method including the step ofcontacting the dopaminergic neurons with a survival-promoting amount ofa substantially purified MANF polypeptide.

In a seventh aspect, the invention features a method for growingdopaminergic neurons for transplantation. This method includes the stepof culturing the neurons, or progenitor cells thereof, with asurvival-promoting amount of a substantially purified MANF polypeptide.In one embodiment, the MANF polypeptide is administered with apharmaceutically acceptable excipient.

In an eighth aspect, the invention features a method of treating apatient having a disease or disorder of the nervous system. The methodincluding the step of administering to the patient a dopaminergicneuronal survival-promoting amount of a substantially purified MANFpolypeptide.

In a ninth aspect, the invention features a method for preventingdopaminergic neuronal cell death in a mammal. This method includes thestep of administering to the mammal a dopaminergic neuronalsurvival-promoting amount of a substantially purified MANF polypeptide.

In a tenth aspect method of transplanting cells into the nervous systemof a mammal such as a human, including (i) transplanting cells into thenervous system of the mammal; and (ii) administering a dopaminergicneuronal survival-promoting amount of a MANF polypeptide to the mammalin a time window from two to four hours before transplanting the cellsto two to four hours after transplanting the cells.

In an eleventh aspect, the invention features another method oftransplanting cells into the nervous system of a mammal such as a human.This method includes the steps of: (a) contacting the cells with a MANFpolypeptide; and (b) transplanting the cells of step (a) into thenervous system of the mammal. It is desirable that step (a) and step (b)be performed within four hours of each other.

In particular embodiments of the fourth, fifth, sixth, seventh, eighth,ninth, tenth, or eleventh aspect, the MANF polypeptide consists of thesequence of SEQ ID NO: 2, 3, 4, 5, 6, or 7, or consists essentially ofSEQ ID NO: 2, 3, 4, 5, 6, or 7

As demonstrated herein, dopaminergic neurons are, in large part,prevented from dying in the presence of a MANF polypeptide. Dopaminergicneurons of the mesencephalon die in patients having Parkinson's disease.The invention thus provides a treatment of Parkinson's disease. Inaddition, the use of a MANF polypeptide in the treatment of disorders ordiseases of the nervous system in which the loss of dopaminergic neuronsis present or anticipated is included in the invention.

The discovery that MANF is involved in dopaminergic neuronal survivalallows MANF to be used in a variety of diagnostic tests and assays foridentification of dopaminergic neuronal survival-promoting drugs. MANFexpression can also serve as a diagnostic tool for determining whether aperson is at risk for a neurodegenerative disorder. This diagnosticprocess can lead to the tailoring of drug treatments according topatient genotype (referred to as pharmacogenomics), including predictionof the patient's response (e.g., increased or decreased efficacy orundesired side effects upon administration of a compound or drug).

Antibodies to a MANF polypeptide can be used both as therapeutics anddiagnostics. Antibodies are produced by immunologically challenging aB-cell-containing biological system, e.g., an animal such as a mouse,with a MANF polypeptide to stimulate production of anti-MANF by theB-cells, followed by isolation of the antibody from the biologicalsystem. Such antibodies can be used to measure MANF polypeptide in abiological sample such as serum, by contacting the sample with theantibody and then measuring immune complexes as a measure of the MANFpolypeptide in the sample. Antibodies to MANF can also be used astherapeutics for the modulation of MANF biological activity.

Thus, in another aspect, the invention features a purified antibody thatspecifically binds to a MANF polypeptide.

In yet another aspect, the invention features a method for determiningwhether a candidate compound modulates MANF-mediated dopaminergicneuronal survival-promoting activity, including: (a) providing a MANFpolypeptide; (b) contacting the MANF polypeptide with the candidatecompound; and (c) measuring MANF biological activity, wherein alteredMANF biological activity, relative to that of a MANF polypeptide notcontacted with the compound, indicates that the candidate compoundmodulates MANF biological activity. The MANF polypeptide can be in acell or in a cell-free assay system.

In another aspect, the invention features a method for determiningwhether candidate compound is useful for decreasing neurodegeneration,the method including the steps of: (a) providing a MANF polypeptide; (b)contacting the polypeptide with the candidate compound; and (c)measuring binding of the MANF polypeptide, wherein binding of the MANFpolypeptide indicates that the candidate compound is useful fordecreasing neurodegeneration.

In particular embodiments of the foregoing screening methods of thepresent invention, the cell is in an animal and the MANF polypeptideconsists of the sequence of SEQ ID NO: 2, 3, 4, 5, 6, or 7, or consistsessentially of SEQ ID NO: 2, 3, 4, 5, 6, or 7.

The invention also features screening methods for identifying factorsthat potentiate or mimic MANF biological activity. In these screeningmethods for potentiators, the ability of candidate compounds to increaseMANF expression, stability, or biological activity is tested using,standard techniques. A candidate compound that binds to MANF may act asa potentiating agent. A mimetic (e.g., a compound that binds a MANFreceptor) is a compound capable of acting in the absence of a MANFpolypeptide.

By “substantially purified” is meant that a polypeptide (e.g., a MANEpolypeptide) has been separated from the components that naturallyaccompany it. Typically, the polypeptide is substantially purified whenit is at least 60%, by weight, free from the proteins andnaturally-occurring organic molecules with which it is naturallyassociated. Preferably, the polypeptide is at least 75%, more preferablyat least 90%, and most preferably at least 99%, by weight, pure. Asubstantially purified polypeptide may be obtained, for example, byextraction from a natural source (e.g., a neural cell), by expression ofa recombinant nucleic acid encoding the polypeptide, or by chemicallysynthesizing the protein. Purity can be measured by any appropriatemethod, e.g., by column chromatography, polyacrylamide gelelectrophoresis, or HPLC analysis.

A polypeptide is substantially free of naturally associated componentswhen it is separated from those contaminants that accompany it in itsnatural state. Thus, a polypeptide which is chemically synthesized orproduced in a cellular system different from the cell from which itnaturally originates will be substantially free from its naturallyassociated components. Accordingly, substantially purified polypeptidesinclude those which naturally occur in eukaryotic organisms but aresynthesized in E. Coli or other prokaryotes.

By “polypeptide” or “protein” is meant any chain of more than two aminoacids, regardless of post-translational modification such asglycosylation or phosphorylation.

A MANF polypeptide that is part of the invention is one havingdopaminergic neuronal survival-promoting activity (“MANF biologicalactivity”) and encoded by a nucleic acid that either hybridizes at highstringency to a cDNA encoding human MANF (SEQ ID NO: 2, 3, or 4) or issubstantially identical to human MANE. Included in this definition arepro-MANE polypeptide (e.g., human pro-MANF; SEQ ID NO: 2), synthetichuman MANE (SEQ ID NO: 4), peptide domains of human MANF (e.g.,LRPGDCEVCISYLGRFYQDLKDRDV TFSPATIENELIKFCREA; SEQ ID NO: 11;RGKENRLCYYIGATDDAATKIIN EVSKPLAHHIPVEKICEKLKKKDSQICEL; SEQ ID NO: 12 andKYDKQIDLS TVDLKKLRVKELKKILDDWGETCKGCAEKSDYIRKINELMPKY; SEQ ID NO: 13)predicted to have MANF biological activity, and counterpart MANFpolypeptides from species such as mouse (e.g., SEQ ID NO: 5, 6, and 7),cow (FIG. 11A; SEQ ID NO: 14), and pig (FIG. 11B; SEQ ID NO: 15).Specifically excluded from the definition of MANF polypeptides are ARPproteins that contain the arginine-rich amino terminus (e.g., aminoacids 1 to 55 of SEQ ID NO: 1). Thus, human ARP (SEQ ID NO: 1) is notconsidered a MANF polypeptide.

A polynucleotide that is a part of the invention is one encoding a MANEpolypeptide, as defined above. Exemplary polynucleotides arerepresented, for example, by the sequences of SEQ ID NO: 9 and SEQ IDNO: 10.

By “substantially identical” is meant a polypeptide or polynucleotideexhibiting at least 5%, preferably 90%, more preferably 95%, and mostpreferably 97% identity to a reference amino acid or nucleic acidsequence. For polypeptides, the length of comparison sequences willgenerally be at least 16 amino acids, preferably at least 20 aminoacids, more preferably at least 25 amino acids, and most preferably 35amino acids. For polynucleotides, the length of comparison sequenceswill generally be at least 50 nucleotides, preferably at least 60nucleotides, more preferably at least 75 nucleotides, and mostpreferably 110 nucleotides.

Sequence identity is typically measured using sequence analysis softwarewith the default parameters specified therein (e.g., Sequence AnalysisSoftware Package of the Genetics Computer Group, University of WisconsinBiotechnology Center, 1710 University Avenue, Madison, Wis. 53705). Thissoftware program matches similar sequences by assigning degrees ofhomology to various substitutions, deletions, and other modifications.Conservative substitutions typically include substitutions within thefollowing groups: glycine, alanine, valine, isoleucine, leucine;aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine;lysine, arginine; and phenylalanine, tyrosine.

By “high stringency conditions” is meant hybridization in 2×SSC at 40°C. with a DNA probe length of at least 40 nucleotides. For otherdefinitions of high stringency conditions, see F. Ausubel et al.,Current Protocols in Molecular Biology, pp. 6.3.1-6.3.6, John Wiley &Sons, New York, N.Y., 1994, hereby incorporated by reference.

By “compound” or “factor” is meant a molecule having an activity thatpromotes the survival (or, conversely, prevents the death) ofdopaminergic neurons in a standard cell survival assay.

By “composition” is meant a collection of polypeptides, including apolypeptide of the present invention.

By “pharmaceutically acceptable excipient” is meant an excipient,carrier, or diluent that is physiologically acceptable to the treatedmammal while retaining the therapeutic properties of the polypeptidewith which it is administered. One exemplary pharmaceutically acceptablecarrier is physiological saline solution. Other physiologicallyacceptable carriers and their formulations are known to one skilled inthe art and described, for example, in Remington: The Science andPractice of Pharmacy, (20th ed.) ed. A. R. Gennaro A R., 2000,Lippencott Williams & Wilkins.

By a compound having “dopaminergic neuronal survival-promoting activity”is the presence of the compound increases survival of dopaminergicneurons by at least two-fold in a dopaminergic neuronal survival assay(such as the one described herein) relative to survival of dopaminergicneurons in the absence of the compound. The increase in the survival ofdopaminergic neurons can be by at least three-fold, more preferably byat least four-fold, and most preferably by at least five-fold. The assaycan be an in vitro assay or an in vivo assay.

The present invention provides new methods and reagents for theprevention of neuronal cell death. The invention also providespharmaceutical compositions for the treatment of neurological diseasesor disorders of which aberrant neuronal cell death is one of the causes.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration showing the effect of differentamounts of conditioned medium from VMCL-1 cell cultures on the survivalof tyrosine hydroxylase-positive cells (top panel) and MAP2-positivecells (bottom panel).

FIG. 2 is a schematic illustration showing human ARP (SEQ ID NO: 1),human pro-MANF (SEQ ID NO: 2) human MANF (SEQ ID NO: 3), and a synthetichuman MANF lacking the signal sequence (SEQ ID NO: 4).

FIG. 3 is a schematic illustration showing mouse pro-MANF (SEQ ID NO:5), mouse MANF (SEQ ID NO: 6), and a synthetic mouse MANF lacking thesignal sequence (SEQ ID NO: 7

FIG. 4 is a schematic illustration showing the sequence ofpolynucleotides encoding human ARP (SEQ ID NO: 8), human pro-MANF (SEQID NO: 9), and human MANF (SEQ ID NO: 10).

FIG. 5 is a schematic illustration showing the biological activity ofMANF expressed in E. Coli on the survival of tyrosinehydroxylase-positive cells (top panel) and MAP2-positive cells (bottompanel). The lanes are as follows: A1-PCM0 control; A2-VMCL-1 CM (25%);B1-MANF (10 pg/mL); B2-MANF (50 pg/mL); B3-MANF (100 pg/mL); B4-MANF(250 pg/mL); B5-MANF (500 pg/mL); B6-MANE (1 ng/mL); B7-MANF (5 ng/mL);B8-MANF (10 ng/mL); B9-MANF (50 pg/mL); B10-MANF (100 ng/mL).

FIG. 6 is a schematic illustration showing the dose-response curve forMANF expressed and secreted from HEK293 cells. The lanes are as follows:A-control; B1-PCM0 control; B2-buffer control; C1-MANF (100 pg/mL);C2-MANF (250 pg/mL); C3-MANF (500 pg/mL); C4-MANF (1 ng/mL); C5-MANF (10ng/mL); C6-MANF (25 ng/mL); C7-MANF (50 pg/mL). All data were collectedwere DIV3, except for A, which was at DIV 1.

FIG. 7 is a schematic illustration showing the rank order potency ofBDNF, GDNF, and MANF.

FIG. 8 is a schematic illustration showing MANF polyclonal antibodyactivity in western blots of MANF (720 ng).

FIG. 9 is a schematic illustration showing the sensitivity of MANFpolyclonal antibody detection in western blots of MANF. As little as15.6 ng MANF was detectable.

FIGS. 10A-10C are a series of schematic illustrations showing that MANFpolyclonal antibody did not cross-react with BDNF or GDNF. (A) westernblot; (B) silver stain; (C) coomassie blue stain.

FIGS. 11A and 11B are schematic illustrations showing the sequence ofcow (FIG. 11A) and pig (FIG. 11B) pro-MANF. The signal peptide isindicated in bold.

DETAILED DESCRIPTION OF THE INVENTION

We previously discovered that a cell line of mesencephalic origin(termed “VMCL-1”) secretes a factor that, in turn, promotesdifferentiation and survival of dopaminergic neurons. This cell linegrows robustly in a serum-free medium. Moreover, the CM prepared fromthese cells contains one or more dopaminergic neuronal survival factorsthat increase the survival of mesencephalic dopaminergic neurons atleast 3-fold, and promotes their development as well.

We purified, from the VMCL-1 cell line, a protein that we identified tobe ARP. We purified this protein as follows. A 3 L volume of VMCL-1conditioned medium was prepared, and subjected to five sequential stepsof column chromatography. At each purification step, each columnfraction was tested for biological activity in the bioassay referred toabove. An estimate of the effect of each fraction on dopaminergicneuronal survival was done at 24 hour intervals, over a period of fivedays, and rated on a scale of 1-10. After the fifth purification step,the biologically active fraction and an adjacent inactive fraction wereanalyzed by SDS-PAGE. The results of the SDS-PAGE analysis revealed adistinctive protein band in the 20 kDa range in the lane from the activefraction. The “active” band was excised and subjected to tryptic digest,and the molecular mass and sequence of each peptide above backgroundwere determined by mass spectrometry analysis. The following two peptidesequences were identified: DVTFSPATIE (SEQ ID NO: 6) and QIDLSTVDL (SEQID NO: 7). A search of the database identified a match for humanarginine-rich protein and its mouse orthologue. The predicted proteinencoded by the mouse EST sequence is about 95% identical to thepredicted human protein. A search of the rat EST database revealed twosequences, one (dbEST Id: 4408547; EST name: EST348489) havingsignificant homology at the amino acid level to the human and mouseproteins. The full-length rat sequence was not in the GenBank database.Additionally, we discovered that the sequence of the human ARP inGenBank was incorrect. The correct sequence is depicted in SEQ ID NO: 1.

We have discovered that human ARP is cleaved such that the arginine-richamino-terminus is separated from the carboxy-terminus to produce humanpro-MANF (SEQ ID NO: 2). The cleaved carboxy-terminal fragment containsa signal peptide, resulting in the secretion of human MANF (SEQ ID NO:3) from the cell.

Both the secreted form and the unsecreted form of MANF (collectivelyreferred to as MANF polypeptides) have neurotrophic activity and areuseful as neurotrophic factor for the treatment of a neurodegenerativedisease such as Parkinson's Disease and for improving dopaminergicneuronal survival during or following transplantation into a human. MANFpolypeptides can also be used to improve the in vitro production ofneurons for transplantation. In another use, MANF polypeptides can beused for the identification of compounds that modulate or mimic MANF'sdopaminergic neuronal survival-promoting activity. MANF polypeptides canalso be used to identify MANF receptors. Each of these uses is describedin greater detail below.

Identification of Molecules that Modulate MANF Biological Activity

The effect of candidate molecules on MANF-mediated regulation ofdopaminergic neuronal survival may be measured at the level oftranslation by using standard protein detection techniques, such aswestern blotting or immunoprecipitation with a MANF-specific antibody.

Compounds that modulate the level of MANF may be purified, orsubstantially purified, or may be one component of a mixture ofcompounds such as an extract or supernatant obtained from cells (Ausubelet al., supra). In an assay of a mixture of compounds, MANE expressionis measured in cells administered progressively smaller subsets of thecompound pool (e.g., produced by standard purification techniques suchas HPLC or FPLC) until a single compound or minimal number of effectivecompounds is demonstrated to MANF expression.

Compounds may also be directly screened for their ability to modulateMANF-mediated dopaminergic neuronal survival. In this approach, theamount of dopaminergic neuronal survival in the presence of a candidatecompound is compared to the amount of dopaminergic neuronal survival inits absence, under equivalent conditions. Again, the screen may beginwith a pool of candidate compounds, from which one or more usefulmodulator compounds are isolated in a step-wise fashion.Survival-promoting activity may be measured by any standard assay.

Another method for detecting compounds that modulate the activity ofMANF is to screen for compounds that interact physically with MANF.These compounds may be detected by adapting interaction trap expressionsystems known in the art. These systems detect protein interactionsusing a transcriptional activation assay and are generally described byGyuris et al. (Cell 75:791-803, 1993) and Field et al., (Nature340:245-246, 1989). Alternatively, MANF or a biologically activefragment thereof can be labeled with ¹²⁵I Bolton-Hunter reagent (Boltonet al. Biochem. J. 133: 529, 1973). Candidate molecules previouslyarrayed in the wells of a multi-well plate are incubated with thelabeled MANF, then washed; any wells with labeled MANF complex areassayed. Data obtained using different concentrations of MANF can beused to calculate values for the number, affinity, and association ofMANF with the candidate molecules.

Compounds or molecules that function as modulators of MANF dopaminergicneuronal survival-promoting activity may include peptide and non-peptidemolecules such as those present in cell extracts, mammalian serum, orgrowth medium in which mammalian cells have been cultured.

A molecule that modulates MANF expression or MANF-mediated biologicalactivity such that there is an increase in neuronal cell survival isconsidered useful in the invention; such a molecule may be used, forexample, as a therapeutic agent, as described below.

The discovery of MANF as a neurotrophic factor that promotes thesurvival of dopaminergic neurons allows for its use for the therapeutictreatment of neurodegenerative diseases such as Parkinson's disease.

To add a MANF polypeptide to cells in order to prevent neuronal death,it is preferable to obtain sufficient amounts of a recombinant MANFpolypeptide from cultured cell systems that can express the protein. Apreferred MANF polypeptide is human MANF, but MANF polypeptides derivedfrom other animals (e.g., pig, rat, mouse, dog, baboon, cow, and thelike) can also be used. Delivery of the protein to the affected tissuecan then be accomplished using appropriate packaging or administratingsystems. Alternatively, small molecule analogs may be used andadministered to act as MANF agonists and in this manner produce adesired physiological effect.

Gene therapy is another potential therapeutic approach in which normalcopies of the gene encoding a MANF polypeptide (or a polynucleotideencoding MANF sense RNA) is introduced into cells to successfullyproduce the MANF polypeptide. The gene must be delivered to those cellsin a form in which it can be taken up and encode for sufficient proteinto provide effective dopaminergic neuronal survival-promoting activity.

Retroviral vectors, adenoviral vectors, adenovirus-associated viralvectors, or other viral vectors with the appropriate tropism for neuralcells may be used as a gene transfer delivery system for a therapeuticMANF construct. Numerous vectors useful for this purpose are generallyknown (Miller, Human Gene Therapy 15-14, 1990; Friedman, Science244:1275-1281, 1989; Eglitis and Anderson, BioTechniques 6:608-614,1988; Tolstoshev and Anderson, Curr. Opin. Biotech. 1:55-61, 1990;Sharp, The Lancet 337: 1277-1278, 1991; Cometta et al., Nucl. Acid Res.and Mol. Biol. 36: 311-322, 1987; Anderson, Science 226: 401-409, 1984;Moen, Blood Cells 17: 407-416, 1991; Miller et al., Biotech. 7: 980-990,1989; Le Gal La Salle et al., Science 259: 988-990, 1993; and Johnson,Chest 107: 77S-83S, 1995). Retroviral vectors are particularly welldeveloped and have been used in clinical settings (Rosenberg et al., N.Engl. J. Med. 323: 370, 1990; Anderson et al., U.S. Pat. No. 5,399,346).Non-viral approaches may also be employed for the introduction oftherapeutic DNA into the desired cells. For example, a MANF-encodingpolynucleotide may be introduced into a cell by lipofection (Feigner etal., Proc. Natl. Acad. Sci. USA 84: 7413, 1987; Ono et al., Neurosci.Lett. 117: 259, 1990; Brigham et al., Am. J. Med. Sci. 298:278, 1989;Staubinger et al., Meth. Enzymol. 101:512, 1983),asialorosonucoid-polylysine conjugation (Wu et al., J. Biol. Chem.263:14621, 1988; Wu et al., J. Biol. Chem. 264:16985, 1989); or, lesspreferably, micro-injection under surgical conditions (Wolff et al.,Science 247:1465, 1990).

Gene transfer could also be achieved using non-viral means requiringinfection in vitro. This would include calcium phosphate, DEAE dextran,electroporation, and protoplast fusion. Liposomes may also bepotentially beneficial for delivery of DNA into a cell. Although thesemethods are available, many of these are of lower efficiency.

In the constructs described, MANF or pro-MANF cDNA expression can bedirected from any suitable promoter (e.g., the human cytomegalovirus(CMV), simian virus 40 (SV40), or metallothionein promoters), andregulated by any appropriate mammalian regulatory element. For example,if desired, enhancers known to preferentially direct gene expression inneural cells may be used to direct MANF polypeptide expression. Theenhancers used could include, without limitation, those that arecharacterized as tissue- or cell-specific in their expression.

RNA molecules may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5′ and/or 3′ ends of the moleculeor the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept can be extended in all of these molecules by the inclusion ofnontraditional bases such as inosine, queosine, and wybutosine, as wellas acetyl-, methyl-, thio-, and similarly modified forms of adenine,cytidine, guanine, thymine, and uridine which are not as easilyrecognized by endogenous endonucleases.

Another therapeutic approach within the invention involvesadministration of a recombinant MANF polypeptide, either directly to thesite of a potential or actual cell loss (for example, by injection) orsystemically (for example, by any conventional recombinant proteinadministration technique).

An additional embodiment of the invention relates to the administrationof a pharmaceutical composition, in conjunction with a pharmaceuticallyacceptable carrier, for any of the therapeutic effects discussed above.Such pharmaceutical compositions may consist of AMNF polypeptides,antibodies to MANF polypeptides, and/or mimetics and agonists of MANFpolypeptides. The compositions may be administered alone or incombination with at least one other agent, such as stabilizing compound,which may be administered in any sterile, biocompatible pharmaceuticalcarrier, including, but not limited to, saline, buffered saline,dextrose, and water. The compositions may be administered to a patientalone, or in combination with other agents, drugs or hormones.

In one example, a MANF polypeptide is administered to a subject at thesite that cells are transplanted. The administration of the MANFpolypeptide can be performed before, during, or after thetransplantation of the cells. Preferably, the two steps are within aboutfour hours of each other. If desirable, the MANF polypeptide can berepeatedly administered to the subject at various intervals beforeand/or after cell transplantation. This protective administration of theMANF polypeptide may occur months or even years after the celltransplantation.

In addition to its administration to a human or other mammal, a MANFpolypeptide can also be used in culture to improve the survival ofneurons during their production any time prior to transplantation. Inone example, the cells to be transplanted are suspended in apharmaceutical carrier that also includes a survival-promoting amount ofa MANF polypeptide. A MANF polypeptide can also be administered to thecultures earlier in the process (e.g., as the neurons are firstdifferentiating). It is understood that the neurons need not be primarydopaminergic neurons. Neurons (e.g., dopaminergic neurons) that aredifferentiated, either in vitro or in vivo, from stem cells or any othercell capable of producing neurons can be cultured in the presence of aMANF polypeptide during their production and maintenance.

Parenteral formulations may be in the form of liquid solutions orsuspensions; for oral administration, formulations may be in the form oftablets or capsules; and for intranasal formulations, in the form ofpowders, nasal drops, or aerosols.

Methods well known in the art for making formulations are to be foundin, for example, Remington: The Science and Practice of Pharmacy, (20thed.) ed. A. R. Gennaro A R., 2000, Lippencott Williams & Wilkins.Formulations for parenteral administration may, for example, contain asexcipients sterile water or saline, polyalkylene glycols such aspolyethylene glycol, oils of vegetable origin, or hydrogenatednaphthalenes, biocompatible, biodegradable lactide polymer, orpolyoxyethylene-polyoxypropylene copolymers may be used to control therelease of the present factors. Other potentially useful parenteraldelivery systems for the factors include ethylene-vinyl acetatecopolymer particles, osmotic pumps, implantable infusion systems, andliposomes. Formulations for inhalation may contain as excipients, forexample, lactose, or may be aqueous solutions containing, for example,polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may beoily solutions for administration in the form of nasal drops, or as agel to be applied intranasally.

The present factors can be used as the sole active agents, or can beused in combination with other active ingredients, e.g., other growthfactors which could facilitate dopaminergic neuronal survival inneurological diseases, or peptidase or protease inhibitors.

The concentration of the present factors in the formulations of theinvention will vary depending upon a number of issues, including thedosage to be administered, and the route of administration.

In general terms, the factors of this invention may be provided in anaqueous physiological buffer solution containing about 0.1 to 10% w/vpolypeptide for parenteral administration. General dose ranges are fromabout 1 mg/kg to about 1 g/kg of body weight per day; a preferred doserange is from about 0.01 mg/kg to 100 mg/kg of body weight per day. Thepreferred dosage to be administered is likely to depend upon the typeand extent of progression of the pathophysiological condition beingaddressed, the overall health of the patient, the make up of theformulation, and the route of administration.

While human MANF is preferred for use in the methods described herein,MANF has been identified in numerous species, including rat, mouse, andcow. One in the art will recognize that the identification of MANF fromother animals can be readily performed using standard methods. Anyprotein having dopaminergic neuronal survival-promoting activity andencoded by a nucleic acid that hybridizes to the cDNA encoding human ARPis considered part of the invention.

The following examples are to illustrate the invention. They are notmeant to limit the invention in any way.

Example 1 Production and Analysis of VMCL-1 Cells

The VMCL-1 cell line was made as follows. Rat E14 mesencephalic cells,approximately 2-3% of which are glioblasts, were incubated in mediumcontaining 10% (v/v) fetal bovine serum for 12 hours and subsequentlyexpanded in a serum-free medium, containing basic fibroblast growthfactor (bFGF) as a mitogen. After more than 15 DIV, several islets ofproliferating, glial-like cells were observed. Following isolation andpassaging, the cells (referred to herein as VMCL-1 cells) proliferatedrapidly in either a serum-free or serum-containing growth medium.Subsequent immunocytochemical analysis showed that they stained positivefor two astrocytic markers, GFAP and vimentin, and negative for markersof oligodendroglial or neuronal lineages, including A2B5, O4, GalC andMAP2. We have deposited the VMCL-1 cell line with the ATCC (AccessionNo: PTA-2479; deposit date: Sep. 18, 2000).

Serum-free CM, prepared from the VMCL-1 cells, caused increased survivaland differentiation of E14 mesencephalic dopaminergic neurons inculture. These actions arc similar to those exerted by CM derived fromprimary, mesencephalic type-1 astrocytes. The expression ofmesencephalic region-specific genes (e.g., win-1, en-1, en-2, pax-2,pax-5 and pax-8), was similar between VMCL-1 cells and primary, type-1astrocytes of E14 ventral mesencephalic origin. In both, wnt-1 wasexpressed strongly, and en-1 less strongly, supporting an expressionpattern expected of their mesencephalic origin. A chromosomal analysisshowed that 70% of the cells were heteroploid, and of these, 50% weretetraploid. No apparent decline in proliferative capacity has beenobserved after more than twenty-five passages. The properties of thiscell line are consistent with those of an immortalized, type-1astrocyte.

The VMCL-1 cells have a distinctly non-neuronal, glial-like morphology,but lack the large, flattened shape that is typical of type-1 astrocytesin culture. Immunocytochemical analysis demonstrated that they stainedpositive for GFAP and vimentin, and negative for MAP2, A2B5 and O4. Thecells were therefore not of the oligodendrocyte lineage. On the basis ofa negative reaction to A2B5 and their morphological characteristics theywere also not type-2 astrocytes. The classification that is supported bythe immunocytochemical evidence is of type-1 astrocytes, although, asnoted, these cells lack the classical morphological traits of primarytype-1 astrocytes in culture.

Example 2 Action of VMCL-1 CM on E14 Dopaminergic Neurons in Culture

VMCL-1 CM was tested at 0, 5, 20 and 50% v/v, for its ability toinfluence survival and development of E14 mesencephalic dopaminergicneurons in culture. The cultures were primed with 10% fetal bovine serum(FBS) for 12 hours, then grown in a serum-free growth medium thereafter,until they were stained and analyzed after 7 DIV. There was adose-dependent action of the CM on the increased survival ofdopaminergic neurons. The CM increased survival by 5-fold. In contrast,there was no significant increase in non-dopaminergic neuronal survival.The profile of the biological action of this putative factor is quitedifferent from that of CM derived from the B49 glioma cell line, thesource of GDNF (Lin et al., Science 260: 1130-1132).

Example 3 Gene Expression Analysis of VMCL-1 Cells

To further investigate the similarity between the VMCL-1 cell line andprimary cultured astrocytes, we measured the expression of six markergenes characteristic of the mesencephalic region. Analysis of wnt-1,en-1, en-2, pax-2, pax-5, and pax-8 showed that all genes were expressedin both E13 and E14 ventral mesencephalon neural tissue, with theexception of pax-2, which was expressed at E13 but not E14 neuraltissue. Both primary astrocytes and VMCL-1 cells expressed wnt-1 atlevels comparable with those of E13 and E14 ventral mesencephalic neuraltissue. The degree of expression of en-1 was similar in primaryastrocytes and VMCL-1 cells, although at a lower level versus expressionin E13 and E14 ventral mesencephalic tissue. In contrast, en-2, pax-5and pax-8 were not expressed in either primary astrocytes or VMCL-1.Pax-2 was expressed in E13 but not E14 ventral mesencephalon, and inprimary astrocytes, but not in VMCL-1.

Example 4 Chromosomal Analysis of VMCL-1 Cells

Chromosomes were counted in 34 cells. Of these, 9 had a count of 42, thediploid number for rat. Of the 25 cells that were heteroploid, 12/25 or48% were in the tetraploid range. Hyperdiploid (counts of 43-48) andhypodiploid (counts of 39-41) cells each accounted for 20% of thepopulation, while 12% of the cells had structurally rearrangedchromosomes.

The selective action of VMCL-1 CM in increasing the survival ofdopaminergic neurons in culture provides a potential clinical use forthe molecule(s) produced by this cell line. The lack of a toxic actionof VMCL-1 CM at a concentration of 50% v/v indicates that the active,putative neurotrophic factor is not toxic. The action exerted by VMCL-1CM mirrors almost exactly that of CM prepared from mesencephalic,primary type-1 astrocytes (Takeshima et al., J. Neurosci. 14: 4769-4779,1994). A high degree of specificity of the putative factor from VMCL-1for dopaminergic neurons is strongly indicated from the observation thatgeneral neuronal survival was not significantly increased, while thesurvival of dopaminergic neurons was increased 5-fold (FIG. 1). We havedemonstrated that primary type-1 astrocytes express GDNF mRNA, but havenot detected GDNF protein by Western blot in the CM, at a sensitivity of50 pg. Moreover, we have shown that under the present experimentalconditions, the increased survival of dopaminergic neurons mediated byan optimal concentration of GDNF is never greater than 2-fold. Theseobservations alone indicate that the factor responsible for theneurotrophic actions of VMCL-1 CM is not GDNF.

Example 5 Production of Type-1 Astrocyte-Conditioned Medium

E16 type-1 astrocyte CM (10 L) was filtered and applied to a heparinsepharose CL-6B column (bed volume 80 mL) which had previously beenequilibrated with 20 mM Tris-HCl (Mallinckrodt Chemical Co. Paris, Ky.)pH 7.6 containing 0.2 M NaCl. After washing with equilibration buffer,bound proteins were eluted from the column with a linear gradient of 0.2M-2 M NaCl in 20 mM Tris-HCl pH 7.6 (400 mL total volume, flow rate 100mL/hr). Fractions were collected using a Pharmacia LKB fractioncollector and absorbance was measured at 280 nm (Sargent-Welch PU 8600UVNIS Spectrophotometer). A 1 mL aliquot was taken from each fraction,pooled into groups of four (4 mL total volume) and desalted usingCentricon-10® membrane concentrators (Millipore, Bedford, Mass.).Samples were diluted 1:4 in defined medium and bioassayed fordopaminergic activity. Active fractions were pooled (80 mL total volume)and then applied to a G-75 Sephadex® column (70×2.5 cm, PharmaciaBiotechnology Ltd., Cambridge, UK) which had been pre-equilibrated with50 mM ammonium formate pH 7.4. Proteins were separated with the samebuffer (flow rate, 75 mL/hr) and absorbance was measured at 280 nm. A 1mL aliquot was taken from each fraction, pooled into groups of four (4mL total volume), concentrated by lyopholyzation and reconstituted in 1mL distilled water volume. Samples were then diluted 1:4 in definedmedium for dopaminergic bioassay. Those with neurotrophic activity werefurther bioassayed as individual fractions.

An important distinguishing feature of VMCL-1 CM is that it promotespredominantly the survival of dopaminergic neurons, compared with thesurvival of GABAergic, serotonergic, and other neuronal phenotypespresent in the culture. This claim of specificity is also made for GDNF.The results of extensive testing have demonstrated, however, that theVMCL-1-derived compound is not GDNF.

Example 6 Isolation and Purification of a Protein Having DopaminergicNeuronal Survival-Promoting Activity

The purification protocol was performed as follows. All salts used wereof the highest purity and obtained from Sigma Chemical Co. All bufferswere freshly prepared and filtered via 0.2 μM filter (GP Expressvacuum-driven system from Millipore)

Step 1: Heparin-Sepharose Column Chromatography (4° C.)

Three liters of VMCL-1 conditioned medium was diluted with an equalvolume of 20 mM sodium phosphate buffer, pH 7.2 at room temperature,filtered, and concentrated to 550 mL volume with 5K PREP/SCALE-TFF 2.5ft₂ cartridge (Millipore). The concentrated material was loaded onto a10 mL Heparin-Sepharose column assembled from 2×5 mL HiTrap Heparincolumns (Pharmacia Biotech) and pre-equilibrated with at least 100 mL of10 mM sodium phosphate buffer, pH 7.2 (buffer A). After the loading wascomplete, the column was washed with 100 mL of buffer A. A total of 10fractions were eluted with buffer B (buffer A plus 1 M sodium chloride)in about 3 mL volumes each. A 300 L sample was withdrawn for analysis.

Step 2: Superose 12 Column Chromatography (4° C.)

All of the fractions from step 1 were pooled, then concentrated to 4.5mL using Centricon Plus-20 concentrator (5,000 MWCO, Millipore), loadedonto 16×600 mm gel-filtration column packed with Superose 12 media (PrepGrade, Sigma. Chemical Co.) and pre-equilibrated with at least 300 mL of20 mM sodium phosphate buffer, pH 7.2 containing 0.6 M sodium chloride(GF buffer). The protein elution was conducted in GF buffer. Twomilliliter fractions were collected and analyzed for activity. Theactive protein was eluted in a 15 mL volume after 84 ml of GF buffer waspassed through the column and corresponded to an approximately 20-30 kDaelution region based on the column calibration data obtained withprotein standards (Bio-Rad).

Step 3: Ceramic Hydroxyapatite Column Chromatography (Room Temperature;FPLC System)

The active fractions from step 2 that corresponded to the 20-30 kDaelution region were pooled and concentrated to 7.5 mL, using a CentriconPlus-20 concentrator (5,000 MWCO), dialyzed overnight at 4° C. against 2L of 10 mM sodium phosphate buffer, pH 7.2 (buffer A) and loaded (viaSuperloop) onto a 1 mL pre-packed ceramic hydroxyapatite (Type I,Bio-Rad) column equilibrated with buffer A. After, the excess of unboundprotein (flow through) was washed off the column with buffer A, thelinear gradient of buffer A containing 1.0 M NaCl was applied from 0 to100%. One milliliter fractions were collected and analyzed for activity.The active protein was eluted as a broad peak within the region ofgradient corresponding to 0.4-0.8 M NaCl concentration.

Step 4: Anion-Exchange Column Chromatography (Room Temperature; FPLCSystem)

The fractions corresponding to the broad peak were pooled (totalvolume=15 mL) and concentrated to 6 mL using Centricon Plus-20 (5,000MWCO), dialyzed overnight at 4° C. against 2 L of 20 mM Tris HCl buffer,pH 7.5 (buffer A), loaded (via Superloop) onto a 1 mL anion-exchangeFPLC column (Uno, Bio-Rad), and equilibrated with buffer A. After theexcess of unbound protein was washed off the column with buffer A, alinear gradient of 0-100% 1 M NaCl (in buffer A) was applied. Onemilliliter fractions were collected and analyzed for activity. Theactive protein was found in the flow-through (i.e., in the unboundprotein fraction).

Step 5: BioSil 125 Column Chromatography (Room Temperature; HPLC System)

The active protein fraction from Step 4 (7 mL of total volume) wasconcentrated down to nearly zero volume (about 1 μL) using CentriconPlus-20 concentrator (5,000 MWCO) and reconstituted in 0.6 mL of 10 mMsodium phosphate buffer, pH 7.2. The reconstituted material (70 μL,analytical run) was loaded onto BioSil 125 HPLC gel-filtration column(Bio-Rad) equilibrated with 20 mM sodium phosphate buffer, pH 7.2 (GFbuffer). The chromatography was conducted using HP 1100 Series HPLCsystem (Hewlett-Packard). The eluate was collected in 120 μL fractionsand analyzed for activity and protein content (SDS-PAGE). The activitywas found in fractions associated with the main 280-nm absorbance peakeluted from the column, which was represented by a 45-kDa proteinaccording to SDS-PAGE analysis. Nevertheless, no activity was found inthe side fractions of the 45-kDa protein peak, indicating that activitymight be due to the presence of another protein that was co-eluted with45 kDa protein, but at much lower concentration that could not bedetected on the 12% SDS-PAGE silver-stained gel. Therefore, theremaining concentrated material from step 5 was further concentrateddown to 80 μL volume using a Centricon-3 concentrator (Millipore), and60 μL was loaded and separated on the column at the same conditions asfor the above-described analytical run. Aliquots of 8 μL were taken fromeach 120 μL fraction of the eluate and analyzed by SDS-PAGE (12% gel)combined with silver staining. This analysis indicated that another twoadditional proteins (having molecular weights of about 18 and 20 kDa)were associated with the active fractions and co-eluted with the major45-kDa protein. The active fractions were dialyzed against 1 L ofammonium acetate buffer, pH 8.0 (4° C.) and combined to create twoactive pools, P-1 and P-2, such that P-1 contained the 20 kDa proteinand the 45 kDa protein, and P-2 contained the 18 kDa protein and the 45kDa protein. Each pool was dried down on SpeedVac vacuum concentrator(Savant) and separately reconstituted in 15 μL 0.1 M ammonium acetatebuffer, pH 6.9. Aliquots were withdrawn from each sample and assayed foractivity. Additionally, 1 μL aliquots were subjected to 12% SDS-PAGEanalysis followed by silver staining.

The results of the foregoing analysis clearly indicated that P-1, butnot P-2, contained the desired survival-promoting activity. In the nextstep, both P-1 and P-2 were dried on SpeedVac, reconstituted (each) in10 μL of freshly prepared SDS-PAGE reducing sample buffer (Bio-Rad),incubated for one minute in a boiling water bath and loaded onto a 12%SDS-PAGE gel. After electrophoresis was complete, the gel was fixed inmethanol/acetic acid/water solution (50:10:40) for 40 minutes at roomtemperature, washed three times with nanopure water, and stainedovernight with GelCode Blue Stain Reagent (Pierce) at room temperature.After staining was completed, and the GelCode solution was washed offthe gel with nanopure water, the visible protein bands corresponding tothe 45 kDa protein (both P-1 and P-2) and the 20 kDa protein (P-1 only)were excised from the gel with a razor blade. Each gel slice containinga corresponding band was placed in a 1.5 mL microcentrifuge tube untilthe time of in-gel digestion.

Example 7 Analysis of In-Gel Digested Fragments by nESI-MS/MS

The protein gel bands were incubated with 100 mM ammonium bicarbonate in30% acetonitrile (aq.) at room temperature for 1 hour in order to removethe colloidal comassie blue stain. The destaining solution was replaceda number of times until the dye was completely removed. The gel pieceswere then covered with deionized water (˜200 μL) and shaken for 10minutes. The gel pieces were dehydrated in acetonitrile and, afterremoving the excess liquid, were dried completely on a centrifugalevaporator. The gel bands were rehydrated with 20 μL of 50 mM ammoniumbicarbonate, pH 8.3, containing 200 ng of modified trypsin (Promega,Madison, Wis.). The gel pieces were covered with 50 mM ammoniumbicarbonate, pH 8.3 (approximately 50 μL), and were incubated overnightat 37° C. The digest solutions were then transferred to clean eppendorftubes and the gel pieces were sonicated for 30 minutes in 50-100 μL of5% acetic acid (aq). The extract solutions were combined with the digestsolutions and evaporated to dryness on a centrifugal evaporator.

The in-gel digest extracts were first analyzed by matrix-assisted laserdesorption ionization-time of flight mass spectrometry (MALDI-TOFMS)using a Voyager Elite STR MALDI-TOFMS instrument (Applied BiosystemsInc., Framingham, Mass.). The extracts were dissolved in 5 μL of 50%acetonitrile, 1% acetic acid. Dihydroxybenzoic acid was used as thematrix and spectra were acquired in positive ion, reflectron mode.Approximately one fifth of each sample was used for this analysis. Thesespectra provided the masses of the peptides in the digest extracts whichwere then used to search an in-house, non-redundant protein sequencedatabase, a process called peptide mass fingerprinting. The remainder ofthe samples were used for peptide sequencing analysis bynanoelectrospray ionization-tandem mass spectrometry (nESI-MS/MS). Theextracts were first desalted using C18 ZipTips (Millipore) andredissolved in 75% methanol (aq.), 0.1% acetic acid (5 μL).Approximately one half of the samples were loaded into nanoelectrosprayglass capillaries (Micromass). nESI-MS/MS analyses were carried outusing a Q-Star quadrupole time-of-flight hybrid mass spectrometer (PESCIEX, Concord, ON). All MS/MS analyses were carried out in positive ionmode. The collision gas was nitrogen and the collision energy was 40-60eV. MS/MS spectra were typically acquired every second over a period oftwo minutes. The MS/MS spectra were used to search an in-housenon-redundant protein sequence database using partial sequence tags(i.e., only the peptide mass and a few fragment ions are used to searchthe database). If the protein was not identified by this procedure thenthe amino acid sequences of two or more peptides were determined asfully as possible from the MS/MS spectra. These sequences were used tocarry out BLAST searches on NCBI's protein, nucleotide and EST sequencedatabases.

Example 8 Identification of MANF, a Secreted Form of ARP

In order for ARP to be a factor that is responsible at least in part forthe observed neurotrophic activity of VMCL-1 CM, the protein must bereleased from the cell. The predicted amino terminus of ARP has basiccharges, however, a property that would favor retention in the cellnucleus. Nonetheless, we hypothesized that there would also exist asecreted form.

Support for our hypothesis was found in a publication by Goo et al.(Molecules and Cells 9:564-568, 1999), who identified a cDNA encoding anARP-like protein in Drosophila melanogaster while screening a cDNAlibrary using a yeast signal sequence trap technique. The putativeARP-like protein encoded by this cDNA lacks the arginine-rich aminoterminus. Using the SignalP program, we identified a signal peptide(residues 1-22) and a signal peptidase-cutting site between alanine 22and leucine 23 of Drosophila ARP-like protein, providing additionalevidence that Drosophila ARP-like protein is secreted.

Based on the alignment between human ARP and Drosophila ARP-likeprotein, we postulated that human ARP would have a signal sequence andsignal peptidase cutting site. Accordingly, we used the SignalP programto analyze the human ARP lacking the arginine-rich amino terminus (aminoacids 1-55); this polypeptide is now referred to as pro-MANF. In thisexample, the methionine at position 56 is the start codon. The SignalPprogram predicted a signal peptide consisting of residues 1-21 of SEQ IDNO: 2 and a cutting site between alanine 21 and leucine 22, which isconsistent with the results from the analysis of the Drosophila ARP-likeprotein. The predicted cleaved human MANF protein is depicted in FIG. 2and SEQ ID NO: 3. This and other exemplary MANF polypeptides are shownin FIGS. 2 and 3. Exemplary MANF polynucleotides are shown in FIG. 4.

Based in part on our analysis of Drosophila ARP-like protein (GenBankAccession No. AF132912_(—)1) and human ARP, we predict that thetranslation can begin at either the methionine at position 1 or themethionine at position 56 of human ARP. In the latter case, the signalpeptide-containing protein (pro-MANF) is capable of being secreted fromthe cell in the form of MANF, where the protein acts a neurotrophicfactor. Our discovery of the existence of MANF does not, however,preclude an intracellular function for the ARP containing thearginine-rich amino-terminal region.

Example 9 Biological Activity of MANF Expressed in E. Coli

Recombinant protein expression was carried out in E. Coli bacterialcells using pTriEx containing a polynucleotide encoding human MANF (SEQID NO: 3). A total of 4 mg of purified recombinant MANE was obtainedfrom 350 mL of bacterial cell culture, its identity confirmed by massspec sequencing. This protein was tested for its ability to protect DAneurons. As shown in FIG. 5, MANF expressed in E. Coli was capable ofprotecting DA neurons from cell death to the same extent as did theVMCL-1 conditioned medium.

Example 10 Dose-Response for Eukaryotic MANF Expressed in HEK293 Cells

The dose-relationship of human MANF (99% pure, produced in HEK293 cells)versus survival of dopaminergic neurons was tested using a dopaminergiccell culture assay system containing 20% of dopaminergic neurons. E14pregnant rats were killed by CO₂ narcosis. The torso was soaked in 70%EtOH, a laporatomy was performed, and the uterine sac removed andtransferred to a 50 mL tube containing 20 mL cold HBSS, pH 7.4. Eachuterine sac was in turn transferred to a 10-cm petri dish containing 15mL cold HBSS. The fetuses were removed intact, and each brain wasisolated intact and transferred to a new 10-cm petri dish containing 15mL cold HBSS. The medial ventral mesencephalon (VM) at the roof of themesencephalic flexure was dissected to obtain 1.0 mm³ piece of tissue ata packing density of 1.0×10⁵ cells/mm³. The VM tissue was transferred toa 15 mL tube containing 10 mL of cold PCM10. The pooled VM tissue waswashed with PCM10 (DMEM/F12 with 2 mM glutamine, 5 mg/mL insulin, 5mg/mL transferrin, 5 mg/mL sodium selenite; 20 nM progesterone, 30 nMthyroxine, and 10% fetal bovine serum) (three washes), followed by asingle wash in serum-free medium (PCMO; same as PCM10 except that itlacks fetal bovine serum) and digested in 2.0 ml of PCM0 containingpapain (10 U/mL) for 15 minutes, at 37° C. The tissue was then rinsed(3×5 mL) with PCM10, to inactivate the protease activity. Triturationwas done in 2.0 mL of PCMO, using a P-1000 set at 500 μL. The end pointis a milky suspension with no signs of tissue clumps. The dispersedcells were centrifuged (1,000 rpm, 2 min, 4° C.), counted, thenresuspended at a density of 6.25×10⁵ cells/mL in PCM 10. Cell viabilitywas tested at this stage, and was usually >95%.

The cells were plated as microisland (MI) droplets of 25 μL, (1.56×10⁴cells/MI) on 8-well chamber slides, coated with poly-D-lysine. A 25 μLMI droplet occupies an area of 12.5 mm². The average, final, mean celldensity of the MI is therefore 1.25×10⁵ cells/cm². The mean cell densityat the center of the MI is about 2.0×10⁵/cm², falling off to <1.0×10⁴ atthe periphery of the MI. The MIs were incubated at 5% CO₂ at 100%humidity for 45 minutes to allow the cells to attach to the coatedsurface. After attachment, 375 μL of PCM 10 was added to each well, andthe cells serum-primed for 4 hr. At the end of priming, 100% of PCM10was aspirated, and replaced with serum-free, PCM0.

MANF was prepared as follows. Human pro-MANF (SEQ ID NO: 2) was clonedinto a pTriEx expression vector. Recombinant protein expression wascarried out in HEK293 cells. Twenty micrograms of purified recombinantMANF lacking the signal sequence) was obtained from 800 mL of HEK293cell conditioned medium. Its identity was confirmed by mass specsequencing analysis.

Cultures were treated on the first, third and fifth days with theindicated amount of MANF. The cultures were fixed and stained on DIV6 orDIV7, using either the Vector ABC method, or indirectimmunofluorescence.

As early as DIV3, there was a significant difference between thedifferent concentrations of MANF tested (ANOVA, P<0.001) (FIG. 6).Paired comparisons using the Tukey method of analysis, indicated thatMANF at 250 and 500 pg/ml and 1.0 and 10 ng/nL were significantlydifferent from controls (P<0.05).

Example 11 Rank Order of Potency among BDNF, GDNF and MANF

As illustrated in FIG. 7, when three equivalent doses of BDNF, GDNF andMANF were tested, the rank order of potency was: MANF>GDNF>BDNF,indicating that the two lower concentrations of MANF were more selectivefor DA neurons, relative to low doses of BDNF or GDNF. At the highestdose, the rank order of potency was: GDNF>MANF>BDNF. In general, BDNFtended to be the most potent, but least specific for protecting DAneurons. At lower concentrations, MANF tended to be the most selectivein protecting DA neurons.

Example 12 Domains of MANF Predicted to be Active

We have identified three peptides from MANF that we predict may haveMANF biological activity (i.e., protect DA neurons from cell death). Thehuman peptides are LRPGDCEVCISYLGRFYQDLKDRDVTFSPATIENELIKFCREA; SEQ IDNO: 11; RGKENRLCYYIGATDDAATKIINEVSKPLAHHIPVEKIC E KLKKKDSQICEL; SEQ IDNO: 12 and KYDKQIDLSTVDLKKLRVKELKKILDDWGETCKGCAEKSDYIRKINELMPKY; SEQ IDNO: 13. Counterpart peptides can be readily identified using standardsequence alignment programs. MANF sequences for mouse, cow, and pig areprovided herein. These peptides can be employed in any of thetherapeutic methods described herein, and are expressly considered to be“MANF polypeptides.”

Example 13 Selectivity of Responsiveness to MANF

The ability of MANF to protect neurons from other brain regions wastested. No protection of rat cerebellar granule neurons, nodose sensoryneurons, or sympathetic noradrenergic neurons was observed. Similarly,in ventral mesencephalic cultures, there appeared to be no activity onGABAergic and serotonergic neurons in cultures in which MANF wasdemonstrably protective for DA neurons. In contrast to the foregoingresults, MANF was protective for a subset of dorsal root ganglion cellsin culture. Dorsal root ganglia consist of at least threesub-populations of neurons. It has been demonstrated that NGF, BDNF andNT-3, all members of the neurotrophin family of neurotrophic factors,each acts on a different subset of these neurons. The action of MANF onthis subset of dorsal root ganglion neurons, is therefore in keepingwith the general neuroprotective proterties of neurotrophic factors.

Example 14 Production of MANF Polyclonal Antibodies

Polyclonal antibodies were prepared as follows. His-tagged full lengthMANF was prepared in E. Coli. Six antigen injections of 200 μg ofpurified MANF protein per injection per rabbit were performed (one eachon days 1, 21, 35, 49, 63, and 70). The serum was collected on day 84(100 mL serum/rabbit).

Western blot analysis was used to test the activity of MANF-pAb. Arelatively high quantity of MANF (720 ng) was used for the initial testof the activity of MANF-pAb, which remained active at a dilution of1:12,800 (FIG. 8; lane 9). In the next test, the dilution of MANF wasfixed at 5,000 and the quantity of MANF varied from 1,000 to 15.6 ng.The lowest quantity of MANF, 15.6 ng, was easily detected (FIG. 9, lane9). In tests for cross reactivity with BDNF and GDNF, the results showedthat even at three times the quantity of MANF (32 ng), the MANF-pAb didnot cross react with either BDNF or GDNF (FIGS. 10A-10C).

The foregoing results were obtained with the following methods.

Mesencephalic Cultures

Primary mesencephalic cell culture was prepared from timed-pregnantSprague-Dawley rats (Taconic Farms; Germantown, N.Y.). as describedpreviously (Shimoda et al., Brain Res. 586:319-323, 1992; Takeshima etal., J. Neurosci. 14:4769-4779, 1994; Takeshima et al., Neuroscience.60:809-823, 1994; Takeshima et al., J. Neurosci. Meth. 67:27-41, 1996).The dissected tissue was collected and pooled in oxygenated, cold (4°C.), HBSS or medium containing 10% fetal bovine serum (Bio fluidsLaboratories, Rockville, Md.), depending on the purpose of theexperiment. Pregnant rats were killed by exposure to CO₂ on thefourteenth gestational day (i.e., E14), the abdominal region was cleanedwith 70% EtOH, a laparotomy was performed, and the fetuses collected andpooled in cold Dulbecco's phosphate-buffered saline (DPBS), pH 7.4,without Ca²⁺ or Mg²⁺. The intact brain was then removed, a cut was madebetween the diencephalon and mesencephalon, and the tectum slit mediallyand spread out laterally. The ventral, medial 1.0 mm³ block of tissuecomprising the mesencephalic dopaminergic region was isolated. Dissectedtissue blocks were pooled in cold (4° C.), oxygenated medium. The tissuewas triturated without prior digestion. Alternatively, the cells wereincubated in L-15 growth medium containing papain (Sigma Chemical Co.),10 U/mL, at 37° C., for 15 minutes, washed (3×2 mL) with medium, andtriturated using only the needle and syringe. The dispersed cells weretransferred to 1.5 mL Eppendorf tubes (1.0 mL/tube), and centrifuged at˜600 g for 2 minutes. The use of higher centrifugation speeds for longerperiods results in contamination of the cultures with debris and, as aresult, suboptimal growth of the cells. The medium was carefullyaspirated, and the cells resuspended in fresh medium and counted using ahemocytometer. All procedures, from laparotomy to plating were completedwithin 2 hours. In a typical experiment, one litter of 10-15 fetusesyielded 1.0×10⁵ cells/fetus, or 1.0×10⁶-1.5×10⁶ cells/litter.

Mesencephalic Microisland Cultures

To make mesencephalic microisland cultures, cells were prepared asdescribed above, and resuspended at a final density of 5.0×10⁵ mL. A 25uL droplet of the suspension (1.25×10⁴ cells) was plated using a 100 μLpipette onto 8-well chamber slides coated with poly-D-lysine (50 μg/mL).The area of the droplet was ˜12.5 mm², for a final mean cell density of1.0×10⁵/cm². The droplet was dispensed uniformly, and the pipette tipwithdrawn vertically, to avoid smearing. The area occupied by themicroisland culture remained uniform for the duration of the culture.The cultures were incubated for 30 minutes at 37° C., in 5% CO₂ at 100%humidity, to allow the cells to attach, and 375 μL of growth medium wasthen added to each well. The medium was changed after the first 12hours, and approximately half of the medium was changed every second daythereafter.

Cell Viability Assay

A two-color fluorescence cell viability assay kit (Live/DeadViability/Cytotoxicity Assay Kits, #L-3224, Molecular Probes, Inc.,Eugene, Oreg.) was used to identify live and dead cells prior to platingand in cultures. Live and dead cells fluoresced green and red,respectively, giving two positive indicators of viability. Ethidiumhomodimer and calcein-AM were diluted with DPBS to give finalconcentrations of 3.8 μM and 2.0 μM, respectively. Evaluation of cellviability was done before plating. A cell suspension was incubated for15 minutes with an equal volume of dye (typically 20 μL) on glass slidesat room temperature in a dark, humid chamber, coverslipped, and thenexamined with a fluorescent microscope using FITC optics. Cell viabilityjust before plating was about 95%.

Culture

The serum-free medium used consisted of equal volumes of Dulbecco'smodified Eagle medium (DMEM) and Ham's F-12 (Gibco, Grand Island, N.Y.;320-1320AJ), 1.0 mg/mL bovine albumin fraction V (Sigma Chemical Co.;A4161), 0.1 μg/mL apo-transferrin (Sigma; T-7786), 5 μg/mL insulin(Sigma; 1-1882) 30 nM L-thyroxine (Sigma; T-0397), 20 nM progesterone(Sigma; P-6149), 30 nM sodium selenite (Sigma; S-5261), 4.5 mM glutamine(Gibco, 320-5039AF), 100 U/mL penicillin, and 100 μg/mL streptomycin(Gibco; P-100-1-91).

Preparation of Conditioned Medium from VMCL-1 Cell Line

In preparing conditioned medium from the VMCL-1 cell line, 2.0×10⁶ cellswere plated in a 15 cm uncoated culture dish, in 20 mL of growth mediumcontaining 1.0% of FBS. At 80% confluence, the medium was aspirated andthe cells washed once with serum-free medium. 20 mL of serum-free N2medium without albumin was added, and conditioning allowed to continuefor 48 hours. During this time, the cells usually expanded to 100%confluence. The medium was aspirated, pooled in 50 mL tubes, centrifuged(15,000 rpm for 20 minutes) and subsequently pooled in a 1.0 L plasticbottle. Usually 5 mL of each batch of CM was filter-sterilized using a0.22 μm filter, stored at aliquots of 5 mL, at −70° C., and used todetermine neurotrophic potency, before being pooled with the largerstore of CM. If desired, VMCL-1 CM can be made in large quantities usingstandard industrial cell culture techniques known to those in the art.

Production of Conditioned Medium for Type-1 Astrocytes

Type-1 astrocytes were prepared as follows. E16 rat fetal brain stem wasdissected in cold DPBS, and the mesencephalic region transferred toastrocyte culture medium (DMEM/Ham's F-12, 1:1, 15% FBS, 4.0 mMglutamine, 30 nM sodium selenite, penicillin, and streptomycin). Cellswere dispersed by trituration in 2 mL of fresh medium using an 18-gaugeneedle fitted to a syringe. Cells were centrifuged for 5 minutes at2,000 rpm in a centrifuge, re-suspended in medium, and triturated again.The final cell pellet was dispersed and plated at a density of 1×10⁶cells/75 cm² flask in 15 mL of medium. Cells were incubated at 37° C.atmosphere of 5% carbon dioxide and 95% air for 24 hours, and unattachedcells were removed by aspiration. Cells were cultured for an additionalnine days, and flasks were then shaken vigorously for 16 hours to removeany contaminating cell types. Astrocyte monolayers were washed threetimes with DPBS, trypsinized and replated (density of 1×10⁶cells/flask). At this time, a small proportion of the cells were platedonto eight-well chamber slides (Nunc Inc., Naperville, Ill.); thesesister cultures were treated as described for the flask cultures. Atconfluence, the medium was replaced with medium containing 7.5% FBS andcells were incubated for 48 hours. At the next exchange, definedserum-free medium (DMEM/Ham's F-12, 1:1, 4.0 mM glutamine, 30 nM sodiumselenite, penicillin 100 U/ml and streptomycin 100 U/mL) was added andcells were incubated for a further 48 hours. Medium was replaced and,after five days, conditioned medium was harvested and mixed withleupeptin (10 mM: Bachem, Torrance, Calif.) and4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (1.0 mM: ICNBiochemicals, Aurora, Ohio) to inhibit proteolysis. At the time ofharvesting, astrocyte monolayers cultured on chamber slides wereimmunostained in order to assess the culture phenotype.

Culturing of VMCL-1 Cells

In culturing VMCL-1 and preparing VMCL-1 CM, 2.0×10⁶ cells were platedin a 15-cm uncoated culture dish, in 20 mL growth medium initiallycontaining 10% FBS. At 80% confluence, the medium was aspirated and thecells washed once with serum-free medium. Usually 20 mL of serum-freemedium without albumin was added, and conditioning allowed to continuefor 48 hours. N2 medium proved to be particularly suitable for use tocollect conditioned medium. During these 48 hours, the cells usuallyexpanded to 100% confluence. The medium was aspirated, pooled in 50 mLtubes, centrifuged (15,000 rpm, 20 min) and pooled in a 1.0 L plasticbottle. Approximately 5 mL of each batch of CM was sterilized using a0.22 mm filter, stored at aliquots of 0.5 mL, at −70° C., and used todetermine neurotrophic potency, before being pooled with the largerstore of CM. The VMCL-1 cell line has now been passaged greater than 50times.

Immunocytochemistry

For MAP2 and TH immunocytochemistry, the cultures were washed (2×250 μL)with cold DPBS, fixed with 4% formaldehyde in PBS for 10 minutes,permeabilized using 1% CH₃COOH/95% EtOH at −20° C., for 5 minutes, andthen washed (3×250 μL) with PBS. Non-specific binding was blocked with1% bovine serum albumin in PBS (BSA-PBS) for 15 minutes. Anti-THantibody (50 μL) (Boehringer-Mannheim, Indianapolis, Ind.), or anti-MAP2antibody (Boehringer-Mannheim) was applied to each well, and the slidesincubated in a dark humid box at room temperature for 2 hours. Controlstaining was done using mouse serum at the same dilution as the anti-THantibody. After washing (2×250 μL) with PBS, anti-mouse IgG-FITC (50 μL)was applied, and the slides incubated for an additional 1 hour. Afterwashing with PBS (2×250 μL), excess fluid was aspirated, the chamberwalls removed, and a single drop of VectaShield mounting medium (VectorLaboratories, Burlingame, Calif.) applied, followed by a cover glass,which was sealed with nail polish. In some experiments, TH wasidentified using biotinylated, secondary antibodies, and thenickel-enhanced, diaminobenzidine (DAB) reaction product was developedusing the biotinylated peroxidase-avidin complex (ABC kit; VectorLaboratories).

For glial fibrillary acidic protein (GFAP, Boehringer-Mannheim,#814369), fixation and permeabilization were done in one step using 5%CH₃COOH/95% C₂H₅OH at −20° C. The subsequent procedures were the same asthose used to visualize TH. For A2B5 and O4, the cultures were washedwith cold DPBS (2×250 μL) and blocked with 1% BSA-PBS for 10 minutes.The A2B5 antibody (50 μL) applied to each well, and incubated for 1hour. After washing with DPBS (2×250 μL), the secondary antibody,anti-IgM-FITC, was applied for 30 minutes. The cells were then washedwith DPBS (2×250 μL). To counter-stain cell nuclei, cells were incubatedwith 0.5 μg/mL of nucleic acid dye H33258 (Hoechst, Kansas City, Mo.) in10 mM sodium bicarbonate for 15 minutes at room temperature, then rinsedin PBS for 2×10 minutes. After a final washing with cold DPBS (2×250μL), they were mounted as described above.

RT-PCR Analysis

Total RNA was extracted from rat E13 or E14 ventral mesencephalic tissueor from 1×10⁹ astrocytes or from 1×10⁹ VMCL-1 cells using RNA-STATreagent (TelTest, University of Maryland, Baltimore, Md.). First strandcDNA was generated from RNA and amplified by polymerase chain reactionusing the manufacturer's procedures.

Reaction products were resolved by 2% agarose gel electrophoresis todetermine size and relative abundance of fragments. PCR results forb-actin and GAPDH were included as controls to confirm equal loading ofcDNA.

Chromosomal Analysis

The cells were grown in DMEM/F-12 1:1 medium supplemented with 2.5% FBS,D-glucose (2.5 g/L) and ITS supplement, diluted 1:100. Twenty-four hourslater, subcultures at metaphase stage were arrested with colchicine (10μg/mL). The cells were trypsinized and subjected to hypotonic shock (75mM KCl). The cells were then fixed in three changes of MeOH/CH₃COOH,3:1, and air-dried. The cells were then stained using 4% Geisma, andmicroscopically examined.

Deposit

Applicant has made a deposit of at least 25 vials containing cell lineVMCL-1 with the American Type Culture Collection, Manassas Va., 20110U.S.A., ATCC Deposit No. PTA-2479. The cells were deposited with theATCC on Sep. 18, 2000. This deposit of VMCL-1 will be maintained in theATCC depository, which is a public depository, for a period of 30 years,or 5 years after the most recent request, or for the effective life ofthe patent, whichever is longer, and will be replaced if it becomesnonviable during that period. Additionally, Applicant has satisfied allthe requirements of 37 C.F.R. §§1.801-1.809, including providing anindication of the viability of the sample. Applicant imposes norestrictions on the availability of the deposited material from theATCC. Applicant has no authority, however, to waive any restrictionsimposed by law on the transfer of biological material or itstransportation in commerce. Applicant does not waive any infringement ofits rights granted under this patent.

Other Embodiments

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will he apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention should not be unduly limited to such specific embodiments.Indeed, various modifications of the described modes for carrying outthe invention which are obvious to those skilled in molecular biology orrelated fields are intended to be within the scope of the invention.

1. A substantially purified polypeptide comprising the sequence of SEQID NO: 2, 11, 12, or
 13. 2. (canceled)
 3. The substantially purifiedpolypeptide of claim 1, wherein said polypeptide consists of thesequence of SEQ ID NO:
 2. 4. A pharmaceutical composition comprising:(i) a substantially purified polypeptide comprising the sequence of SEQID NO: 2; and (ii) a carrier that is pharmaceutically acceptable foradministration to the central nervous system.
 5. The pharmaceuticalcomposition of claim 4, wherein said polypeptide consists essentially ofthe sequence of SEQ ID NO:
 2. 6. A pharmaceutical composition consistingof the sequence of SEQ ID NO: 11, 12, or
 13. 7. The pharmaceuticalcomposition of claim 4, further comprising a neural cell.
 8. Thepharmaceutical composition of claim 7, wherein said neural cell is aneuron, a neural stem cell, or a neuronal precursor cell. 9.-33.(canceled)